Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, has a non-segmented, negative-sense, single-stranded RNA genome. Its eleven kb genome has five genes which encode five structural proteins of the virus; the nucleocapsid protein (N), which is required in stoichiometric amounts for encapsidation of the replicated RNA; the phosphoprotein (P), which is a cofactor of the RNA-dependent RNA polymerase (L); the matrix protein (M) and the attachment glycoprotein (G) (e.g., see Gallione et al. 1981, Rose and Gallione, 1981; Rose and Schubert, 1987 and Schubert et al., 1985; U.S. Pat. Nos. 6,033,886; 6,168,943).
VSV is an arthropod borne virus that can be transmitted to a variety of mammalian hosts, most commonly cattle, horses, swine and rodents. VSV infection of humans is uncommon, and in general is either asymptomatic or characterized by mild flu-like symptoms that resolve in three to eight days without complications. Because VSV is not considered a human pathogen, and pre-existing immunity to VSV is rare in the human population, the development of VSV derived vectors has been a focus in areas such as immunogenic compositions and gene therapy. For example, studies have established that VSV can serve as a highly effective vector for immunogenic compositions, expressing influenza virus haemagglutinin (Roberts et al., 1999), measles virus H protein (Schlereth et al., 2000) and HIV-1 env and gag proteins (Rose et al., 2001). Other characteristics of VSV that render it an attractive vector include: (a) the ability to replicate robustly in cell culture; (b) the inability to either integrate into host cell DNA or undergo genetic recombination; (c) the existence of multiple serotypes, allowing the possibility for prime-boost immunization strategies; (d) foreign genes of interest can be inserted into the VSV genome and expressed abundantly by the viral transcriptase; and (e) the development of a highly specialized system for the rescue of infectious virus from a cDNA copy of the virus genome (U.S. Pat. Nos. 6,033,886; 6,168,943).
Although there is little evidence of VSV neurological involvement during natural infection, animals (e.g., primates, rodents, herd animals) that are inoculated intracerebrally (and in the case of rodents intranasally) with wild-type virus, mouse brain passaged wild-type virus or cell culture adapted wild-type virus, can develop clinical signs of disease, and usually die two to eight days post inoculation. Because of these observations, and the need to produce a vector for immunogenic compositions for use in humans that has an exceptional safety profile, VSV vectors under development are tested in stringent, primate and small animal neurovirulence models. These tests are designed to detect any residual virulence in attenuated VSV vectors before consideration for advancement to human clinical trials.
The attenuation of prototypic-VSV vectors resulted from the accumulation of multiple nucleotide substitutions throughout the virus genome during serial passage in vitro and the synthesis and assembly of the genome cDNA. These mutations had pleiotropic effects that rendered the virus less pathogenic in mice than the lab-adapted virus from which it was derived (e.g., see Roberts et al., 1998). Prototypic further attenuated VSV vectors were also developed by truncation of the cytoplasmic tail region of the virus G protein, leading to VSV mutants that were defective in budding from the plasma membrane of infected cells (Schnell et al., 1998).
Currently known VSV vectors, putatively attenuated or not, have had unacceptable levels of residual virulence when tested in small animal and non-human primate neurovirulence models. The development of a VSV vector for uses such as a vector for immunogenic compositions, a gene therapy vector and the like, will require VSV vectors having minimal to non-detectable levels of pathogenicity in animal neurovirulence models. Thus, there is presently a need in the art of viral vectors to identify genetically modified, attenuated VSV mutants having significantly reduced (or eliminated) pathogenicity in mammals.